A Preliminary Study On Screening Of The Porcine Circovirus Type 2 Resistant Genes By Capsid Targeted Vrial Inactivation

Porcine circovirus type 2 (PCV2) is the primary pathogen of post-weaning pigs multisystemic wasting syndrome (PMWS), and is also intimately associated with Porcine dermatitis and nephropathy syndrome (PDNS), Porcine respiratory disease complex (PRDC), Proliferative and necrotizing Pneumonia (PNP), congenital tremor AII (CT-AII) and etc . All these diseases are called PCV2 associated disease. PCV2 associated disease, as a sort of severe immunosuppressive disease, has given rise enormous economic loss in pig industry.In the face of the enormous loss in pig industry, there is no yet corresponding effective vaccine and druggery at the market. So it has become the present research hotspots to quest for new strategies in anti-PCV2 infection. Capsid targeted viral inactivation (CTVI) is a newly emerging protein-based antiviral strategy. Compared with other nucleic-acid-based antiviral strategies, CTVI is more steady, specific and can efficiently avoid the immune escape resulted by pathogen mutation. The emergence of CTVI provided a new train of thought in confronting PCV2 infection.To explore the feasibility of CTVI in anti-PCV2, we constructed the capsid targeted nucleases fusion gene and examined its antiviral effect.We directly amplified and obtained the coding sequence of staphylococcal nuclease from staphylococcus aureus; and got two kinds of capsid protein fusion gene via recombination, one of which has staphylococcal nuclease gene at amino terminal (JNPC), the other one at carboxyl terminal(PNJC). The fusion gene has been expressed in E. coli. using pET-20b(+) as expression vector, and the nuclease activity of the fusion protein has been examined. Results showed that the molecular masses of two constructed fusion proteins are both about 47KD, just as expected. Western Blot analysis with specific antibodies showed that the products are target fusion proteins. The products have the nuclease activity, capable of cutting and degrading nucleic acid. These results laid the foundation for further investigating the effect of CTVI strategy on anti-PCV2 infection.The eukaryotic expression cassette for JNPC, PNJC and Cap gene respectively had been constructed using pIRESneo as the vector. After cell transfection and G418 screening, steadly expression PK15 cell lines for the three genes mentioned above had been obtained (pIRESneo transgenic cell line as negative control). Then the integration and expression status of foreign genes in these transgenic cell lines had been anlaysed through PCR, RT-PCR, Northern Blot and Western Blot which showed that the steadly expression cell line for the genes mentioned above had been obtained and the fousion protein genes being expressed at a relatively lower level for abnormal splicing of the fusion genes. The fusion proteins can be detected by Western Blot and they show good nuclease activity in DNA digestion test. All these indicate that these cell lines can be used in the next experiment to verify the feasibility of CTVI in anti-PCV2.The four cell lines mentioned above and the untransgenic cells all experienced the virus passage experiment, then the PCV2 genome content had been measured for the anti-viral effect analysis, which showed that after passage of 10 generations, compared with the control group, the virus titer of JNPC transgenic cell line reduced about 60.55â„… and 53.46â„…, respectively via semi-quantitative PCR and fluorescence quantitative PCR, with the virus titers between other control groups extremely approximate. The good accordance of the two quantitative PCR results has confirmed the accuracy of the data. This result preliminarily bore out the applicability of CTVI strategy in screening the PCV2 resistant genes. We obtained the capsid protein fusion gene JNPC with a degree of antiviral effect. But compared with other applications of CTVI in virology, this result is not desirable enough yet. The antiviral efficiency is relatively lower.In conclusion, we have borne out the applicability of CTVI strategy in treatment of PCV2 infection and laid the foundation for screening PCV2 resistant gene and subsequentaly breeding anti-PCV2 transgenic pigs.